Engineering Yeast for Efficient and Robust Incorporation
of Selenomethionine into Expressed Proteins

One major drawback to the use of yeast for preparation of proteins for structural analysis by crystallographic methods is the lack of an effective method for incorporation of selenomethionine.  Selenomethionine-substituted proteins are widely used for phasing of diffraction patterns by multiwavelength anomalous dispersion (MAD) phasing methods.  This methodology is well established in E. coli, in which selenomethionine incorporation into proteins is robust and effective, but is not well established in any other organism.  In yeast, efficient selenomethionine incorporation into expressed proteins has been difficult because of the toxicity of selenomethionine in yeast. 

Drs. Elizabeth Grayhack and Eric Phizicky at the University of Rochester considered that selenomethionine toxicity is due to its conversion to Se-adenosyl-selenomethionine, and therefore engineered a yeast strain to block this metabolic step.  Consistent with this hypothesis, they first showed that sam1-D sam2-D double mutant strains (which lack both of the yeast S-adenosylmethionine synthetases) can grow on medium containing selenomethionine, whereas wild type strains or sam1-D or sam2-D single mutants are sensitive to selenomethionine.  Second, they showed that Ncl1 protein and each of 6 other tested proteins were efficiently produced in sam1-D sam2-D mutant strains in medium containing 0.5 mM selenomethionine, but were not expressed in the corresponding wild type strains in this medium.  Third, they demonstrated by mass spectrometry that selenomethionine was incorporated at 95% efficiency into yeast Ncl1 protein expressed in the sam1-D sam2-D mutant yeast strain in media containing 0.5 mM selenomethionine, and at 90 % in media containing 0.25 mM selenomethionine.  Fourth, they expressed and purified Wrs1 protein (tryptophanyl-tRNA synthetase) in media containing 0.25 mM selenomethionine, and Dr. Mike Malkowski and colleagues at the Hauptman-Woodward Institute crystallized the protein and successfully determined its crystal structure by MAD phasing.

These results constitute a general solution to the problem of effective selenomethionine incorporation into proteins expressed in yeast, thus removing a major obstacle for the use of yeast as an alternative to E. coli for expression of proteins for structural analysis by X-ray crystallography.  Furthermore, these results also suggest that efficient selenomethionine incorporation in other organisms might be effected by the employment of similar methods to prevent conversion of selenomethionine into Se-adenosylselenomethionine.  These finding were recently published in Proceedings of the National Academy of Sciences, U. S. A.

Reference

Malkowski, M. G., Quartley, E., Friedman, A. E., Babulski, J., Kon, Y., Wolfley, J., Said, M., Luft, J. R., Phizicky, E. M., DeTitta, G. T., and Grayhack, E. J. (2007) Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc. Natl. Acad. Sci. U S A. 104:6678-6683. [PubMed]